Over the past decade there has been a tremendous increase in the number of reports aimed at elucidating the role of astrocytes in brain physiology. The results of these studies are consistent with the possibility that the activity of astrocytes is tightly coupled to that of neurons. However, the vast majority of these studies have utilized cultures of astroglia prepared from immature CNS tissue and grown in the absence of their normal cellular and chemical milieu. Although these studies have been very important in characterizing the properties of cultured astroglia that may be important in vivo, very little progress has been made in establishing what role (if any) these properties actually play in the developing and mature CNS. The reason why we and so many others have relied on primary cultures of astroglia is fairly obvious: techniques have not been available for studying astrocytes in the living brain. It is our premise that in order to firmly establish the role of astrocytes in brain physiology, it will be necessary to develop new methods whereby the astroglial properties that have been described using cultured cells can be assessed in vivo. The overall and long term goal of this project is to understand the mechanisms and degree of neuronal-glial communication in developing and mature CNS. The importance of this work lies not in the receptors and neurotransmitters studied but in the different roles for astrocytes suggested by either dynamic communication between neurons and astrocytes or the lack of such communication. Recent findings support the hypothesis that immature astrocytes require a large number of different receptor- signalling systems to respond to their changing neuronal environment during development and that many (possibly most) of these are lost with maturation of the CNS. This hypothesis suggests that the degree to which the function of mature astrocytes is influenced by neurotransmitters is much less than suggested from in vitro studies. A major goal of this proposal is to determine whether the ability of immature astrocytes to sense neuronal activity changes during their differentiation into mature astrocytes in vivo. Confocal microscopy together with fluorescent ion sensitive probes will be used to examine the influence of neurons on astroglial responsiveness in vitro and the ability of immature and mature astrocytes in vivo to respond to neuronal activity. Furthermore, we will examine the hypothesis that the activation of astrocytic receptors serve, in part, to modulate gap junction communication between astrocytes. This hypothesis stems from recent findings in this laboratory which indicate the astroglial receptors linked to the activation of protein kinase C (PKC) inhibit gap junction communication between these cells. Overall, the results of these studies should determine whether neuronal-astrocytic communication via neurotransmitters if predominantly present in the immature CNS or widespread in the adult CNS. These findings would have a major impact on our view of the role of astrocytes in brain physiology.